VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins (original) (raw)
Figure 5
Metals do not affect VPS29 phosphatase activity or interaction with VPS35.
(A) SDS-PAGE gel showing purified VPS29 and trimeric retromer proteins used for phosphatase assays stained with Coomassie Blue. (B) No detectable phosphatase activity was measured for VPS29 alone or in complex with VPS35 and VPS26. Phosphatase assays used the CI-MPR peptide CSSTKLVSFHDD(pS)DEDLLHI. The release of phosphate was measured using Biomol Green reagent and colorimetric assay at 620 nm. Calf intestinal alkaline phosphatase (CIAP) is shown for comparison. (C) When VPS29 has bound metal, the conformation of Phe63 is altered such that it may clash with VPS35 and inhibit binding. The diagram shows a close up of the interaction between VPS29 and VPS35 [24]. The Mn2+-bound VPS29 structure (green ribbon, and yellow side-chains) is overlayed with VPS35-bound VPS29 (blue ribbon and cyan side-chain). VPS35 is shown in surface representation. (D) No significant difference is observed in binding to VPS35 in the presence of EDTA or MnCl2 indicating that metals do not influence complex formation. VPS29 interaction with VPS35 was analysed by ITC; top panels show raw data and bottom panels show integrated normalised data.